Reactive oxygen species mediated DNA damage in human lung alveolar epithelial (A549) cells from exposure to non-cytotoxic MFI-type zeolite nanoparticles

Increasing utilization of engineered nanoparticles in the field of electronics and biomedical applications demands an assessment of risk associated with deliberate or accidental exposure. Metal based nanoparticles are potentially most important of all the nanoparticles in terms of health risks. Microporous alumino-silicates and pure silicates named as zeolites and zeo-type materials with variety of structures, chemical compositions, particle sizes and morphologies have a significant number of industrial uses such as in catalysis, sorption and ion-exchange processes. In particular, the nanosized particles due to their unique properties are used in hybrid organic-inorganic materials for photography, photonics, electronics, labeling, imaging, and sensing. The aim of the current study is to investigate pure silica MFI-type zeolites nanoparticles with sizes of 50 nm and 100 nm (samples MFI-50 and MFI-100) under suspended conditions and their toxicological effects on human lung alveolar (A549) cells under in vitro conditions.     

Ciclesonide uptake and metabolism in human alveolar type II epithelial cells (A549)

Background

Ciclesonide is a novel inhaled corticosteroid for the treatment of airway inflammation. In this study we investigated uptake and in vitro metabolism of ciclesonide in human alveolar type II epithelial cells (A549). Ciclesonide uptake was compared with fluticasone propionate, an inhaled corticosteroid that is not metabolized in lung tissue. A549 cells were incubated with 2 × 10^-8 M ciclesonide or fluticasone propionate for 3 to 30 min to determine uptake; or with 2 × 10^-8 M ciclesonide for 1 h, followed by incubation with drug-free buffer for 3, 6, and 24 h to analyze in vitro metabolism. High performance liquid chromatography with tandem mass spectrometry was used to measure the concentrations of both corticosteroids and metabolites.

Results

At all time points the mean intracellular concentration was higher for ciclesonide when compared with fluticasone propionate. Activation of ciclesonide to desisobutyryl-ciclesonide (des-CIC) was confirmed and conjugates of des-CIC with fatty acids were detected. The intracellular concentration of ciclesonide decreased over time, whereas the concentration of des-CIC remained relatively stable: 2.27 to 3.19 pmol/dish between 3 and 24 h. The concentration of des-CIC fatty acid conjugates increased over time, with des-CIC-oleate being the main metabolite.

Conclusion

Uptake of ciclesonide into A549 cells was more efficient than that of the less lipophilic fluticasone propionate. Intracellular concentrations of the pharmacologically active metabolite des-CIC were maintained for up to 24 h. The local anti-inflammatory activity of ciclesonide in the lung may be prolonged by the slow release of active drug from the depot of fatty acid esters.     

Nanoparticles attenuate P-glycoprotein/MDR1 function in A549 human alveolar epithelial cells

P-glycoprotein/MDR1 (P-gp) is a well-characterised membrane transporter relevant in drug disposition and multi-drug resistance. In this study, we aimed to investigate how far nanoparticulates impair the function of the P-gp transport system and which particle properties govern these interactions.Expression and function of P-gp was confirmed in A549 cell monolayers. Rhodamine 123 (Rh123) release studies were carried out in the presence of known inhibitors of P-gp function (i.e., cyclosporine A and verapamil), under ATP depletion (NaN₃/DOG) and after acute exposure to nanoparticles (NPs) with different surface modifications, ζ-potentials and sizes (plain, carboxylated, and amine- and sulphate-modified). The cytotoxic potential of NPs on A549 monolayers was evaluated by MTT assay. The effects on P-gp protein level, after incubation with NPs, were investigated by Western blot analysis of A549 cell lysate and supernatant.Cellular retention of Rh123 was significantly (P < 0.05) increased in the presence of carboxylated (100 nm), amine- and sulphate-modified NPs. A slight, but not significant, decrease in Rh123 release was also observed for plain latex and carboxylated (500 nm) NPs. The MTT assay demonstrated that most NPs caused only marginal levels of cytotoxicity (78-88% cell viability); the positively charged amine-NPs, however, were considerably more cytotoxic. Western blot showed that NPs did not cause any cell membrane disruption.Our findings suggest that nanomaterials can attenuate membrane transporter function depending on their size and surface properties and hence might influence the disposition of xenobiotics as well as endogenous substrates.     

Aflatoxin G1-induced oxidative stress causes DNA damage and triggers apoptosis through MAPK signaling pathway in A549 cells

Our previous studies showed that Aflatoxin G₁ (AFG₁) could induce lung adenocarcinoma, and that the cancer cells originated from alveolar type II cells (AT-II cells). Recently, we found AFG₁ induced structural impairment in rat AT-II cells, which may account for an early event in lung tumorigenesis. However, the mechanism of AFG₁-induced AT-II cell damage remains unclear. DNA damage and apoptosis induced by oxidative stress are well accepted causes of cell damage. Thus, we explore whether AFG₁ activates the reactive oxygen species (ROS)/MAPK/apoptosis pathway to cause cell damage in human AT-II cells like the cell line (A549). We found AFG₁ induced oxidative stress by increasing ROS generation and caused DNA double-strand breaks (DSBs) by up-regulating γH2AX expression. AFG₁ also triggered apoptosis in A549 cells by regulating Fas/FasL, caspase-8, Bax, Bcl-2, and activating caspase-3. Pre-treatment with antioxidant n-acetyl-l-cysteine (NAC) reduced ROS generation and DNA DSBs, inhibited apoptosis, and increased cell viability in AFG₁-treated cells. Furthermore, we found AFG₁ activated ROS-mediated JNK and p38 pathways to induce cell apoptosis in A549 cells. In conclusion, our results indicate that AFG₁ induces oxidative DNA damage and triggers apoptosis through ROS-mediated JNK and p38 signaling pathways in A549 cells, which may contribute to AFG₁-induced AT-II cell damage.     

The role of mitogen-activated protein kinases in crystalline silica-induced cyclooxygenase-2 expression in A549 human lung epithelial cells

We examined the role of mitogen-activated protein kinase (MAPK) signaling pathways in crystalline silica-induced expression of cyclooxygenase (COX)-2, an important mediator of airway inflammation, in A549 human lung epithelial cells. The levels of COX-2 mRNA increased after a 30-min exposure, and COX-2 protein increased after a 2-h exposure to crystalline silica. Both remained elevated at 8?h; however, no change was observed in the expression of the constitutive COX-1 isoform. The level of prostaglandin E₂, a major product of COX enzymes, increased in response to crystalline silica exposure. Phosphorylated forms of MAPKs including extracellular signal-regulated protein kinase (ERK), c-Jun NH₂-terminal kinase, and p38 were also increased after crystalline silica exposure. COX-2 expression was markedly suppressed by treatment with the p38 inhibitor, SB203580, and mildly suppressed by the MAPK/ERK kinase inhibitor, U0126. Treatment with the nuclear factor-κB (NF-κB) inhibitor, BAY11-7082, markedly suppressed silica-induced COX-2 expression. These results show that crystalline silica exposure induces COX-2 expression in A549 cells in a manner that is dependent on the MAPK and NF-κB pathways. Although a marked induction of MAPK phosphatase (MKP)-1 expression was observed in A549 cells exposed to crystalline silica, the silencing of MKP-1 expression using short interference RNA did not affect silica-induced COX-2 expression, suggesting that the down-regulation of COX-2 expression by MKP-1 is unlikely.     

Mitoxantrone-loaded superparamagnetic iron oxide nanoparticles as drug carriers for cancer therapy: Uptake and toxicity in primary human tubular epithelial cells

Superparamagnetic iron oxide nanoparticles (SPIONs) are in use for many clinical diagnostic and experimental therapeutic applications, for example, for targeted drug delivery. To analyze the cellular responses to mitoxantrone-carrying SPIONs (SPION-MTO), and to the drug released from SPIONs, we used an in vitro system that allows comparison of primary human cells with different endocytotic capacities, namely, epithelial cells from proximal and distal parts of the nephron. SPIONs were selectively and rapidly internalized by proximal tubular cells with high endocytotic potential, but not by distal tubular cells. Uptake did not affect cell viability or morphology. In both cell types, free MTO (10–100?nM) induced double-strand DNA breaks and senescence, cell hypertrophy and reduced cell proliferation. However, cadherin-mediated cell–cell adhesion, cytoskeletal structures or polarity of the cells were not affected. Interestingly, a comparable response was also observed upon treatment with SPION-MTO and was independent of uptake of the particles. The effect of SPION-MTO on cells which did not internalize particles was primarily related to the release of MTO from drug-coated particles upon incubation in serum-containing cell growth medium. In conclusion, we show that whereas the uptake of SPIONs does not affect cellular functions or viability, the toxicity of drug-loaded SPIONs depends essentially on the type of drug bound to nanoparticles. Due to the relatively low systemic toxicity of MTO, the effects of MTO-SPIONs on human tubular cells were moderate, but they may become clinically relevant when more nephrotoxic drugs are bound to SPIONs.     

人肺泡上皮细胞株A549和脐静脉内皮细胞中的内皮素-1通路(英文)

AIM: This study was designed to characterize the en-dothelin pathway in an immortalized human adenocarcino-ma-derived alveolar epithelial cell line (A549) and human umbilical vein endothelial cell line (HUVEC). METHODS: The release of BT-1 and big-ET-1 was measured in the incubation medium of both cell lines. The expression of mRNAs coding for the endothelin iso-forms (hppET-1, -2, -3), the endothelin converting enzymes (hECE-la, b, c, and d) and the hETA and hETB receptors was investigated using RT-PCR. The expression of ECE-1 mRNA in various human tissues and in A549 cells was investigated by Northern blot analysis and the subcellular localization of ECE-1 in A549 cells was investigated by immunoblotting using a polyclonal antibody. RESULTS: Under control conditions, HUVEC release both ET-1 and big-ET-1 (ratio 5 to 1) while in A549 cells the big-ET-1 levels were below the threshold of detection. The release of these two peptides was minimally affected by various inhibitors of peptidases. However, in b     

Alleviating effects of reduced graphene oxide against lead-induced cytotoxicity and oxidative stress in human alveolar epithelial (A549) cells

Broad application of reduced graphene oxide (rGO) and ubiquitous lead (Pb) pollution may increase the possibility of combined exposure of humans. Information on the combined effects of rGO and Pb in human cells is scarce. This work was designed to explore the potential effects of rGO on Pb-induced toxicity in human alveolar epithelial (A549) cells. Prepared rGO was polycrystalline in nature. The formation of a few layers of visible creases and silky morphology due to high aspect ratio was confirmed. Low level (25 μg/mL) of rGO was not toxic to A549 cells. However, Pb exposure (25 μg/mL) induced cell viability reduction, lactate dehydrogenase enzyme leakage with rounded morphology in A549 cells. Remarkably, Pb-induced cytotoxicity was significantly mitigated by rGO co-exposure. Pb-induced mitochondrial membrane potential loss, cell cycle arrest and higher activity of caspase-3 and -9 enzymes were also alleviated by rGO co-exposure. Moreover, we observed that Pb exposure causes generation of pro-oxidants (e.g., reactive oxygen species, hydrogen peroxide and lipid peroxidation) and antioxidant depletion (e.g., glutathione and antioxidant enzymes). In addition, the effects of Pb on pro-oxidant and antioxidant markers were significantly reverted by GO co-exposure. Inductively coupled plasma-mass spectrometry suggested that due to the adsorption of Pb on rGO sheets, accessibility of Pb ions for A549 cells was limited. Hence, rGO reduced the toxicity of Pb in A549 cells. This research warrants further study to work on detailed underlying mechanisms of the mitigating effects of rGO against Pb-induced toxicity on a molecular level.     

Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models

Abstract

We explored how to assess the genotoxic potential of nanosize particles with a well validated assay, the in vitro cytochalasin-B micronucleus assay, detecting both clastogens and aneugens. Monodisperse Stöber amorphous silica nanoparticles (SNPs) of three different sizes (16, 60 and 104 nm) and A549 lung carcinoma cells were selected as models. Cellular uptake of silica was monitored by ICP-MS. At non-cytotoxic doses the smallest particles showed a slightly higher fold induction of micronuclei (MNBN). When considering the three SNPs together, particle number and total surface area appeared to account for MNBN induction as they both correlated significantly with the amplitude of the effect. Using nominal or cellular dose did not show statistically significant differences. Likewise, alkaline comet assay and FISH-centromeric probing of MNBN indicated a weak and not statistically significant induction of oxidative DNA damage, chromosome breakage and chromosome loss. This line of investigation will contribute to adequately design and interpret nanogenotoxicity assays.     

Genotoxic and oxidative stress effects of 2-amino-9H-pyrido[2,3-b]indole in human hepatoma G2 (HepG2) and human lung alveolar epithelial (A549) cells

Abstract

2-Amino-9H-pyrido[2,3-b]indole (AαC), which is present in high quantities in cigarette smoke and also in fried food, has been reported to be a probable human carcinogen. However, few studies have reported on the genotoxicity and oxidative stress induced by AαC. This study investigated the genotoxic effects of AαC in human hepatoma G2 (HepG2) and human lung alveolar epithelial (A549) cells using the comet assay. Significant increases in DNA fragment migration indicated that AαC causes serious DNA damage in HepG2 and A549 cells. The role of oxidative stress in the mechanism of AαC-induced genotoxicity was clarified by measuring the level of intracellular reactive oxygen species (ROS), the GSH/GSSG ratio and the formation of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage. The results showed that the levels of ROS and 8-OHdG increased, whereas the GSH/GSSG ratio decreased. The concentration of 8-OHdG was positively related to DNA damage. Taken together, these results indicate that AαC can induce genotoxicity and oxidative stress and that AαC likely exerts genotoxicity in HepG2 and A549 cells through ROS-induced oxidative DNA damage. This is the first report to describe AαC-induced genotoxic and oxidative stress in HepG2 and A549 cells.     

Concentration‐dependent cytokine responses of silica nanoparticles and role of ROS in human lung epithelial cells

Reactive oxygen species (ROS) is regarded as a critical denominator in nanoparticle toxicology and inflammation. Previously, we have shown that silica nanoparticles sized 50 nm (Si50) induce release of CXCL8 and IL‐6 from BEAS‐2B cells, via mechanisms involving NFκB, p38 MAP kinase and TGF‐α‐activated EGF receptor. In the present study, the role of ROS‐mediated mechanisms in the concentration‐dependent Si50 induction of CXCL8 and IL‐6 responses was examined. Si50 (200 µg/mL) induced a time‐dependent ROS formation and a postponed increase in expression of haem oxygenase (HO‐1) mRNA and protein. Pre‐treatment with the ROS inhibitors N‐acetyl cysteine (NAC) and diphenyleneiodonium (DPI) partially attenuated CXCL8 and IL‐6 responses to 200 µg/mL, but not to 100 µg/mL Si50. The release of TGF‐α induced by Si50 (200 µg/mL) was significantly reduced by NAC, but not by DPI nor siRNA against NADPH oxidase DUOX‐1 (siDUOX‐1). Furthermore, siDUOX‐1 reduced Si50‐induced CXCL8, but not IL‐6. Both p38 and p65 phosphorylations were inhibited by siDUOX‐1, but for NAC only p65 phosphorylation reached a significant reduction. Neither NAC nor DPI reduced Si50‐induced CXCL8 and IL‐6 gene expressions. In conclusion, Si50‐induced CXCL8 and IL‐6 involved both ROS‐dependent and ROS‐independent mechanisms. Notably, the role of ROS seemed restricted to effects of higher concentrations of Si50 and not mediated via the gene expression.     

Hydroxyapatite nanoparticle‐induced mitochondrial energy metabolism impairment in liver cells: in vitro and in vivo studies

Hydroxyapatite nanoparticles (HAP‐NPs) have been extensively developed as drug carriers, bone implants, coating materials, etc. in the human body. However, research focusing on the potential side effects of HAP‐NPs on the mitochondria‐associated energy metabolism in liver cells is lacking. In this study, HAP‐NPs with a long diameter of 80 nm and a short diameter of 20 nm were evaluated for their ability to induce mitochondrial energy metabolism dysfunction in vitro and in vivo . In the in vitro system, the buffalo rat hepatocyte (BRL) cell line was directly exposed to the HAP‐NPs. The results of these experiments showed that the HAP‐NPs induced inhibition of mitochondrial dehydrogenase activity, which was accompanied by a decrease in the mitochondrial membrane potential (MMP). In addition, HAP‐NPs elevated the hepatic levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and decreased the levels of GSH and SOD. These data indicated that HAP‐NPs induced a lowered rate of electron transfer in the mitochondrial respiratory chain, accompanied by a decrease in the activity of the mitochondrial respiratory chain complexes I, II and III. Furthermore, HAP‐NPs induced a decline in the enzymatic expression in the Krebs cycle. We also investigated the role of Kupffer cells (KCs, rat‐derived) in the effects induced by the HAP‐NPs. The supernatant from the HAP‐NP‐treated KCs was used to stimulate the BRL cells. We observed that the HAP‐NPs had the ability to induce KC activation. The activation of KCs then led to the release of tumor necrosis factor‐α (TNF‐α), nitric oxide (NO) and reactive oxygen species (ROS), and induced the inhibition of mitochondrial respiratory chain complexes I, II and III in the BRL cells. In the in vivo study, the TEM examination revealed mitochondrial swelling and vacuolar degeneration in the HAP‐NP‐treated hepatocytes. In addition, the amount of succinate (Suc), an intermediate in the mitochondrial Krebs cycle, also declined in the ¹H NMR spectroscopic measurements. Copyright © 2017 John Wiley & Sons, Ltd.     

Suppression of zinc-induced p53 phosphorylation and p21 expression by wortmannin in A549 human pulmonary epithelial cells

In A549 cells treated with zinc sulfate (ZnSO₄), the levels of p53 phosphorylated at Ser15 and total p53 protein increased. Treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K)-related kinases, suppressed ZnSO₄-induced phosphorylation and accumulation of p53 protein. Expression of cyclin-dependent kinase inhibitor p21, one of the genes regulated by p53, was up-regulated following exposure to ZnSO₄, and suppressed by preincubation with wortmannin. These results suggest that zinc might induce the phosphorylation of p53 at Ser15 through wortmannin-sensitive pathway(s) at least in part, and result in the transactivation of the p21 gene in this human pulmonary epithelial cell line.     

Enhancing the efficiency of bortezomib conjugated to pegylated gold nanoparticles: an in vitro study on human pancreatic cancer cells and adenocarcinoma human lung alveolar basal epithelial cells

ABSTRACT

Objectives: Gold nanoparticles have become promising vectors for cancer diagnosis and treatment. The present study investigates the effect of bortezomib (BTZ), a proteasome inhibitor, conjugated with pegylated gold nanoparticles (PEGAuNPs) in pancreatic and lung cancer cells.

Methods: Synthesized gold nanoparticles (PEGAuNPs) were conjugated with bortezomib antitumor drug. We investigated the cytotoxicity induced by BTZ conjugated with functionalized gold nanoparticles in vitro, in the human pancreatic (S2-013) and lung (A549) cancer cell lines.

Results: We found an efficient of conjugation of BTZ with PEGAuNPs. In vitro assays showed that after 72 h’ incubation with PEGAuNPs-BTZ cancer cells revealed alterations in morphology; also for S2-013 and A549 cancer cells, the IC₅₀ value of free BTZ is respectively 1.5 and 4.3 times higher than the IC₅₀ value of PEGAuNPs-BTZ. Furthermore, for TERT-HPNE, the IC₅₀ value is around 63 times lower for free BTZ than the conjugated nanovehicle. Cell growth inhibition results showed a remarkable enhancement in the effect of BTZ when conjugated with AuNPs.

Conclusions: Our findings showed that conjugation with PEGAuNPs enhance the BTZ growth-inhibition effect on human cancer cells (S2-013 and A549) and decreases its toxicity against normal cells (TERT-HPNE).     

Long and short fiber amosite asbestos alters at a different extent the redox metabolism in human lung epithelial cells

The mechanism by which asbestos fibers are fibrogenic and tumorigenic is still matter of debate. The higher pathogenicity of longer fibers has been traditionally associated with their slower clearance in respiratory airways. However, short amosite fibers, obtained by grinding longer ones, exhibited a lower potential to damage nude DNA and a lower in vitro cytotoxicity. We have thus revisited the two sets of long and short fibers in order to compare their surface properties to their activity in cell systems. In this study we report that, in human lung epithelial cells A549, long amosite fibers, more effectively than the short ones, initiate free radical reactions, inhibit the glucose 6-phosphate dehydrogenase activity and the pentose phosphate pathway, decrease the intracellular level of reduced glutathione, and increase the generation of thiobarbituric acid reactive substances and the leakage of lactate dehydrogenase in the extracellular medium. These results suggest that the shortening of fibers by prolonged milling affects not only their biopersistence, but also their surface properties, hence their interaction with cellular metabolism. Our data provide also a mechanism by which asbestos fibers inhibit the pentose phosphate pathway, i.e., via the oxidative inhibition of glucose 6-phosphate dehydrogenase, which is prevented by reduced glutathione.     

Cytotoxicity of carbon nanotube variants: A comparative in vitro exposure study with A549 epithelial and J774 macrophage cells

Abstract

While production of engineered carbon nanotubes (CNTs) has escalated in recent years, knowledge of risk associated with exposure to these materials remains unclear. We report on the cytotoxicity of four CNT variants in human lung epithelial cells (A549) and murine macrophages (J774). Morphology, metal content, aggregation/agglomeration state, pore volume, surface area and modifications were determined for the pristine and oxidized single-walled (SW) and multi-walled (MW) CNTs. Cytotoxicity was evaluated by cellular ATP content, BrdU incorporation, lactate dehydrogenase (LDH) release, and CellTiter-Blue (CTB) reduction assays. All CNTs were more cytotoxic than respirable TiO₂ and SiO₂ reference particles. Oxidation of CNTs removed most metallic impurities but introduced surface polar functionalities. Although slopes of fold changes for cytotoxicity endpoints were steeper with J774 compared to A549 cells, CNT cytotoxicity ranking in both cell types was assay-dependent. Based on CTB reduction and BrdU incorporation, the cytotoxicity of the polar oxidized CNTs was higher compared to the pristine CNTs. In contrast, pristine CNTs were more cytotoxic than oxidized CNTs when assessed for cellular ATP and LDH. Correlation analyses between CNTs’ physico–chemical properties and average relative potency revealed the impact of metal content and surface area on the potency values estimated using ATP and LDH assays, while surface polarity affected the potency values estimated from CTB and BrdU assays. We show that in order to reliably estimate the risk posed by these materials, in vitro toxicity assessment of CNTs should be conducted with well characterized materials, in multiple cellular models using several cytotoxicity assays that report on distinct cellular processes.